Summary

口服联合抗逆转录病毒治疗HIV-1感染人源化小鼠

Published: October 06, 2022
doi:

Summary

该协议描述了一种提供口服联合抗逆转录病毒药物的新方法,该方法成功抑制人源化小鼠中的HIV-1 RNA复制。

Abstract

人类免疫缺陷病毒(HIV-1)大流行继续在世界范围内有增无减地蔓延,目前还没有针对艾滋病毒的疫苗。虽然联合抗逆转录病毒疗法(cART)成功地抑制了病毒复制,但它不能完全根除艾滋病毒感染者的宿主。安全有效的HIV感染治愈策略需要多管齐下的方法,因此HIV-1感染动物模型的进步对于HIV治愈研究的发展至关重要。人源化小鼠概括了HIV-1感染的关键特征。人源化小鼠模型可以被HIV-1感染,并且可以通过cART方案控制病毒复制。此外,cART中断导致人源化小鼠的病毒迅速反弹。然而,对动物进行cART给药可能无效、困难或有毒,并且许多临床相关的cART方案无法得到最佳利用。除了对研究人员来说可能不安全之外,通过常用的强化日常注射程序施用cART还会通过动物的身体约束来引起压力。本文中描述的治疗HIV-1感染人源化小鼠的新型口服cART方法导致病毒血症抑制低于检测水平,增加CD4 +恢复率,并改善HIV-1感染人源化小鼠的整体健康状况。

Introduction

慢性人类免疫缺陷病毒(HIV)感染者的预期寿命通过联合抗逆转录病毒治疗(cART)显著提高12。cART成功地减少了大多数HIV-1慢性感染参与者的HIV-1复制并将CD4 + T细胞计数提高到正常水平3,从而改善了整体健康状况并显着减少了疾病进展4。然而,即使在急性感染期间开始 ART 时,潜伏的 HIV-1 储库也会建立567在 ART 期间,储库持续多年,并且 ART 中断后病毒快速反弹是有据可查的89。接受抗逆转录病毒治疗的艾滋病毒感染者也容易患心血管疾病、癌症和神经系统疾病等合并症的风险更高101112。因此,需要一种功能性的HIV治疗方法。HIV-1感染的动物模型在开发和验证新的HIV治疗策略方面具有明显的优势131415。人源化小鼠作为一种小动物模型,可以在不同组织中提供多谱系人免疫细胞重建,从而可以密切研究HIV感染16171819在人源化模型中,人源化骨髓-肝脏-胸腺(BLT)模型成功地概括了慢性HIV-1感染以及人类对HIV-1感染的功能性免疫反应202122,2324因此,人源化BLT小鼠模型已被广泛用于HIV研究领域的各个方面的研究。人源化BLT小鼠不仅是重现持续HIV-1感染和发病机制的成熟模型,也是评估基于细胞疗法的干预策略的重要工具。目前的作者和其他人已经证明,人源化BLT小鼠模型概括了持续的HIV-1感染和发病机制25,2627,并提供了评估基于细胞疗法的干预策略的工具2829,30,313233

cART方案由每天服用的抗逆转录病毒药物组合组成,可抑制HIV-1复制,以至于成功治疗的个体的病毒载量在长期内仍然无法检测到34。用临床相关的cART方案治疗HIV感染的人源化小鼠的结果类似于在HIV-1感染的ART治疗个体中观察到的结果22:HIV-1水平被抑制在检测限以下,并且cART的中断导致HIV复制从潜伏库反弹35。皮下注射(SC)27,36,37或腹膜内(IP)373839是人源化小鼠cART治疗的常用途径。然而,每天密集的注射会通过身体约束给动物带来压力40。它也是劳动密集型的,并且由于在使用锐器时暴露于艾滋病毒而增加,因此对研究人员来说可能是不安全的。口服给药是模仿HIV-1感染者服用的cART药物的吸收、分布和排泄的理想选择。口服给药通常涉及定制且通常费力的程序,将抗逆转录病毒药物放入灭菌(由于小鼠的免疫缺陷而必需)食物2437,41或水42,43,444546,这可能与许多抗逆转录病毒药物在化学上相容,也可能不相容,或者导致小鼠不容易吃或喝的东西(这会影响体内的剂量和药物水平)。这里提出的新型经口cART给药方法超越了以前的给药尝试,因为它与不同类型的抗逆转录病毒药物相容,安全性和易于制备和给药,以及减少每日注射引起的动物压力和焦虑。

富马酸替诺福韦二吡呋酯 (TDF)、艾维特拉韦 (ELV) 和拉替拉韦 (RAL) 是水溶性较差的药物。有趣的是,在高脂肪食物中观察到TDF的生物利用度增加,这表明脂肪食物对脂肪酶的竞争性抑制可能为TDF47提供一定的保护。因此,选择DietGel Boost杯代替普通啮齿动物食物作为交付方法,基于其适度的脂肪含量(每100克20.3克),与普通啮齿动物食物(每100克10克10克)和典型的小鼠高脂肪饮食(每100克40-60克)相比48。一杯的总重量为75克;因此,每个杯子将包含足够五只小鼠在3天内食用的食物量,因此也包含足够的药物。

Protocol

匿名的人类胎儿组织是通过商业获得的。动物研究是根据加州大学洛杉矶分校和(加州大学洛杉矶分校)动物研究委员会(ARC)根据所有联邦,州和地方指南批准的协议进行的。具体而言,所有实验均按照美国国立卫生研究院(NIH)和国际实验动物护理评估和认证协会(AALAC)关于实验动物住房和护理的建议和指南进行,根据加州大学洛杉矶分校ARC协议编号2010-038-02B。所有手术均在氯胺酮(100mg / …

Representative Results

假设一只体重为25克的平均小鼠每天消耗4克食物,则通过口服摄入的每日药物剂量对应于2.88mg / kg TFV,83mg / kg FTC和768mg / kg RAL。为了测试与每日注射cART相比,优化的食物方案是否有毒并影响整体健康,在cART之前和期间通过口服或皮下注射每周监测小鼠体重。每组在cART给药前没有显着的体重差异(图1)。然而,在每日cART SC注射期间,小鼠重量不断下降。相比之下,DietGel中?…

Discussion

本文开发了一种口服cART给药方法,用于HIV-1感染的人源化小鼠,方法是在高营养食物中结合三种抗逆转录病毒药物。与每日注射给药相比,口服给药更易于使用,限制给药频率,减少动物处理,最大限度地减少压力并提高安全性55。到目前为止,只有少数针对人源化小鼠243741的研究使用含有粉碎ART药物的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢Romas Geleziunas博士和Jeff Murry博士以及吉利德的人们提供本研究中使用的抗逆转录病毒药物。这项工作由NCI 1R01CA239261-01(到厨房),NIH资助P30AI28697(加州大学洛杉矶分校CFAR病毒学核心,基因和细胞治疗核心,以及人源化小鼠核心),U19AI149504(PIs:Kitchen&Chen),CIRM DISC2-10748,NIDA R01DA-52841(到Zhen),NIAID R2120200174(PIs:Xie&Zhen),IRACDA K12 GM106996(Carrillo)。这项工作还得到了加州大学洛杉矶分校艾滋病研究所,詹姆斯·B·彭德尔顿慈善信托基金和麦卡锡家庭基金会的支持。

Materials

60 mm petri dish Thermo Scientific Nunc 150288 For aliquoting ART food
APC anti-human CD8 Antibody Biolegend 344722 For flow cytometry
BD LSRFortessa BD biosciences For flow data collection
CD34 microbeads Miltenyi Biotec 130-046-702 For NSG-BLT mice generation
Centrifuge tubes Falcon 14-432-22 For dissolving ART
DietGel Boost ClearH2O 72-04-5022 For making ART food
Elvitegravir Gilead Gifted from Gilead
Emtricitabine Gilead Gifted from Gilead
FITC anti-human CD3 Antibody Biolegend 317306 For flow cytometry
Flowjo software FlowJo For flow cytometry data analysis
HIV-1 forward primer: 5′-CAATGGCAGCAATTTCACCA-3′; IDT Customized For viral load RT-PCR
HIV-1 probe: 5′-[6-FAM]CCCACCAACAGGCGGCCT
TAACTG [Tamra-Q]-3′;
IDT Customized For viral load RT-PCR
HIV-1 reverse primer: 5′-GAATGCCAAATTCCTGCTTGA-3′; IDT Customized For viral load RT-PCR
Human fetal tissue Advanced Bioscience Resources, Inc
Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ The Jackson Laboratory 5557 For constructing the humanized mice
Pacific Blue anti-human CD45 Biolegend 304022 For flow cytometry
PerCP anti-human CD4 Antibody Biolegend 300528 For flow cytometry
QIAamp Viral RNA Kits Qiagen  52904 For measuring viral load
Raltegravir Merck Gifted from Merck
Sterile cell scrapers Thermo Scientific 179693 For aliquoting ART food
TaqMan RNA-To-Ct 1-Step Kit Applied Biosystems 4392653 For plasma viral load detection
Tenofovir disoproxil fumarate Gilead Gifted from Gilead
Trimethoprim-Sulfamethoxazole Pharmaceutical Associates NDC 0121-0854-16 For keeping ART food sterile. Each 5mL teaspoon contains
200 mg Sulfamethoxazole, USP
40 mg Trimethoprim, USP
NMT 0.5% Alcohol

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Cite This Article
Mu, W., Zhen, A., Carrillo, M. A., Rezek, V., Martin, H., Lizarraga, M., Kitchen, S. Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice. J. Vis. Exp. (188), e63696, doi:10.3791/63696 (2022).

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